Molecular mechanisms of botulinum neurotoxin-induced neurite outgrowth in motor neurons derived from stem cells
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Botulinum neurotoxin serotype A (BoNT/A), known to produce neuromuscular paralysis (botulism) with extremely high potency is also a well-established therapeutic agent used to alleviate the pain and dysfunction associated with a number of neurologic and muscular disorders. Independent of the cellular mechanism that causes botulism, BoNT/A has been found to promote neurite outgrowth of motor neurons. The cellular mechanism(s) underlying this phenomenon remain unknown. In this dissertation, we use HBG3 embryonic stem cell-derived motor neurons (MNs) as an in vitro model to explore mechanisms of BoNT/A-induced neurite outgrowth. The results showed first that the pharmacological inhibitor U0126, which blocks activation of the extracellular signal-regulated kinases 1/2 (ERK1/2), reduced total neurite length and secondary branch formation, while primary neurite formation was not significantly altered. Brain-derived neurotrophic factor (BDNF) enhanced ERK1/2 phosphorylation and promoted secondary neurite formation and increased total neurite length, without changing primary neurite numbers. These findings indicate that BDNF induced ERK1/2 activation is one pathway mediating neurite development in HBG3-derived MNs. BoNT/A stimulated primary and secondary branch formation as well as total neurite length in HBG3-MNs, similar to previously published findings for mouse primary MNs. The phosphorylation of ERK1/2 was enhanced transiently in response to BoNT/A. Blocking ERK1/2 activation reduced the stimulatory effects of BoNT/A, whereas blocking the activity of AKT or classical protein kinase C isoforms did not. Moreover, RT-PCR microarray analyses revealed that bdnf expression was elevated in response to BoNT/A exposure in cultured embryonic primary MNs, and quantitative PCR confirmed the upregulation of bdnf in HBG3-MNs. Inhibition of the ERK1/2 pathway blocked the BoNT/A-induced bdnf upregulation. The BDNF scavenger TrkB-Fc showed nearly complete blockade of the stimulatory effect of BoNT/A on primary and secondary neurite formation, as well as total neurite length. Taken together, these data suggest that ERK1/2 signaling is critical to mediate secondary neurite branching during development, particularly when stimulated by the neurotrophin BDNF. BoNT/A induces secondary neurite formation through enhancing ERK1/2 activity to up-regulate bdnf expression, while the mechanism(s) for basal and toxin-stimulated primary branch formation is not fully determined.